Tissue culture allows the study in vitro of animal cell behavior in an investigator-controlled physiochemical environment. However, cellular morphology and metabolic activity of cultured cells are affected by the composition of the substrate on which they are grown. Presumably cultured cells function best (i.e. proliferate and perform their natural in vivo functions) when cultured on substrates that closely mimic their natural environment. Currently, studies in vitro of cellular function are limited by the availability of cell growth substrates that present the appropriate physiological environment for proliferation and development of the cultured cells.
The ability of complex substrates to support cell growth in vitro has been previously reported, and matrix products supporting such growth are commercially available. For example, Becton Dickinson currently offers two such products: Human Extracellular Matrix and MATRIGEL.RTM. Basement Membrane Matrix. Human Extracellular Matrix is a chromatographically partially purified matrix extract derived from human placenta and comprises laminin, collagen IV, and heparin sulfate proteoglycan. (Kleinman, HK et al., U.S. Pat. No. 4,829,000 (1989)) MATRIGEL.RTM. is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor, gelled to form a reconstituted basement membrane. Both of these matrix products require costly biochemical isolation, purification, and synthesis techniques and thus production costs are high.
The present invention is directed to the use of vertebrate submucosa-derived matrices as substrates for the growth and attachment of a wide variety of cell types. The collagenous matrices for use in accordance with the present invention comprise highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. The extracellular collagenous matrix for use in this invention is derived from submucosal tissue of a warm-blooded vertebrate. Submucosal tissue can be obtained from various sources, including intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebrates. This tissue can be used in either its natural configuration or in a comminuted or partially digested fluidized form. Vertebrate submucosal tissue is a plentiful by-product of commercial meat production operations and is thus a low cost cell growth substrate, especially when the submucosal tissue is used in its native layer sheet configuration.
The submucosal cell growth substrates of this invention provide cells with a collagenous matrix environment in vitro resembling that found in vivo. The natural composition and configuration of submucosal tissue provides a unique cell growth substrate that promotes the attachment and proliferation of cells.
Accordingly, one object of the present invention is to provide a relatively inexpensive cell culture growth substrate that promotes or induces growth and differentiation of cells cultured in vitro.
Another object of this invention is to provide a method for improving cell proliferation in cell/tissue culture by using vertebrate submucosal tissue as a substrate for cell/tissue growth in vitro.
Another object of this invention is to provide a cell culture composition including a proliferating cell population in contact with submucosal tissue of a warm-blooded vertebrate and a nutrient medium for support of the growth of said cell population.
Still a further object of this invention is to provide a model system for studying tumor cell growth. The model system comprises a proliferating tumor cell population in contact with submucosal tissue of a warm-blooded vertebrate and a nutrient medium. The submucosal tissue substrates provide an in vitro environment resembling that found in vivo and thus in accordance with the present invention serve as a model system for the study of tumor cell growth characteristics. Such a model system would allow detailed characterization of the cellular and molecular processes involved in tumor cell growth and invasion of nontumor tissues.
One other object of this invention is to provide a culture system (referred to herein as an "invasion chamber") and a method for analyzing the invasive growth properties of eukaryotic cells. The invasion chamber comprises first and second chambers separated by a substrate interface, wherein the substrate interface comprises submucosa tissue. Cells are cultured in the invasion chamber by seeding the cells directly onto the submucosal substrate interface, filling the first and second chambers with nutrient media to support the growth of cells, and culturing the cells under conditions conducive to the growth of the cells. The cells can be cultured under optimal growth conditions to study general cell growth characteristics, or the growth conditions can be varied to study the response of cells to those changes in growth conditions.
In one embodiment, tumor cells are cultured in contact with the substrate interface of the invasion chamber under varying growth conditions to investigate the growth and invasive properties of the tumor cells and their response to various growth conditions. The mucosal tissue substrate and the tumor cell population on the substrate can then be examined using standard histological means.
It has been reported that compositions comprising submucosal tissue of the intestine of warm-blooded vertebrates can be used as tissue graft materials in sheet or fluidized form. U.S. Pat. No. 4,902,508 describes tissue graft compositions that are characterized by excellent mechanical properties, including high compliance, a high burst pressure point, and an effective porosity index. These properties allow such compositions to be used for vascular and connective tissue graft constructs. When used in such applications the preferred graft constructs serve as a matrix for the in vivo regrowth of the tissues replaced by the graft constructs. U.S. Pat. No. 5,275,826 describes use of fluidized forms of vertebrate submucosal tissues as injectable or implantable tissue grafts.
An additional object of the present invention is to enhance or expand the functional properties of vertebrate submucosal tissues as an implantable or injectable tissue graft construct by seeding the submucosal tissue in vitro with preselected or predetermined cell types prior to implanting or injecting the graft construct into the host.